Apparatus for directing particles in a fluid

ABSTRACT

There is disclosed apparatus for directing particles entrained in a fluid, comprising a chamber having a first wall, including means for generating a sound wave having a frequency v, and a second, opposite wall capable of reflecting the sound wave in which the first and second wall define a conduit for the passage of the fluid, and in which the thickness of the second wall is such that the path length of the standing wave in the second wall is a multiple of about ½ the wavelength λ r  of the sound wave therein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the U.S. national phase of International ApplicationNo. PCT/GB2003/001125 filed on Mar. 18, 2003 and published in English onMar. 25, 2004 as International Publication No. WO 2004/02487 A1,whichapplication claims priority to Great Britain Application No. 0221391.6filed on Sep. 16, 2002, the contents of which are incorporated byreference herein.

The present invention concerns apparatus for directing particles in afluid. The present invention is particularly, although not exclusively,concerned with apparatus utilising an ultrasound standing wave having asingle pressure node in the fluid to direct particles to a plane surfaceboundary.

It is known that the creation of an ultrasound standing wave in a fluidcomprising a suspension of particles exerts a force on the particles,which acts to direct them towards a pressure node or a pressureantinode. In particular, it is known that bacteria in aqueous suspensionare directed towards pressure nodes on account of the fact that theirmass density and the speed of sound therein are greater than in water.By contrast, oil droplets or air bubbles in water are directed topressure antinodes because their mass density and the speed of soundtherein are lower than in water.

The magnitude and direction of these acoustic radiation forces have beenextensively discussed. See for example, King, L. V. Proc. R. Soc., 1934,London A147, 212-240, Yosioka, K. and Kawasima, Y., Acustica, 1955, 5,167-173, Gor'kov, L. P., Sov. Phys. Dokl., 1962, 6, 773-775, Nyborg, W.L., J. Acoust. Soc. Am., 1967, 42, 947-952, Crum, L. A., ibid., 1971,50, 157-163, Gould, R. K. and Coakley, W. T, Proc. 1973 Symp. onFinite-Amplitude Wave Effects in Fluids (Pergamon Press, Guildford, UK1974), pp 252-257, Whitworth, G. and Coakley, W. T., J. Acoust. Soc.Am., 1992, 91, 79-85 and Gröschl M., Fundamentals Acustica—actaacustica, 1998, 84, 432-447.

It is well known that pressure antinodes in an acoustic standing wavesystem generally occur at a boundary or boundaries between materialshaving significantly different acoustic impedance. For example, pressureantinodes occur at a boundary between water and stainless steel or glasslayers. It has not, until now, been possible to direct particles to aboundary surface by arranging for a pressure node at that surface. Thus,U.S. Pat. Nos. 5,225,089, 5,626,767, EP 0 633 049 and EP 0 380 194 alldisclose acoustic apparatus for directing particles in which a pressureantinode is present at boundary surfaces.

The position of pressure nodes in an ultrasound standing wave system isof particular interest when it is desired to generate a standing wavehaving a single node in the fluid layer. Such systems require that thewidth or thickness of the fluid layer is a half or a quarter of thewavelength λ_(f) of the standing wave in the fluid.

The system comprising a half wavelength thickness, herein referred to asthe λ/2 system, is known to the art and extensively discussed by Hawkes,J. J. et al., in J. Acoust. Soc. Am., 2002, 111, 1259-1266 and Sensorsand Actuators B, 2001, 75, 213-222. An acoustic chamber comprises afirst wall having a piezoceramic and a stainless steel transmissionlayer and a second, reflecting wall of glass. The frequency of the soundwave is selected so that the thickness of the glass reflector is aquarter wavelength of the standing wave in glass in order to ensure themaximum energy in the system is focused in the fluid layer. Pressureantinodes formed at the boundaries of the piezoceramic and steel layer,the steel layer and fluid layer and the fluid layer and glass wall leadto a pressure node for the standing wave at the centre of the fluidlayer. The λ/2 system has been used to filter particles from a fluid(Spengler, J. F. et al., Bioseparation, 2001, 9, 329-341) as well as tomove particles from one fluid to another (see our co-pending GB patentapplication No. GB0223562.0).

The system comprising a quarter wavelength thickness, herein referred toas the λ/4 system and similar to that described above, has previouslybeen thought to be of no practical value. The apparent requirement thatthe frequency of the sound wave is selected so that the thickness of theglass reflector is a quarter wavelength of the standing wave in glasslimits the system. In particular, a pressure node can only occur at theboundary of the fluid layer and one or other of the steel layer or glasswall. However, because a pressure node must also occur at boundariescontacting air, on account of the low acoustic impedance of air, a phasechange may occur in the standing wave system. This phase changeinvariably occurs at the piezoceramic so that a pressure node is formednot at the boundary between the fluid and the glass wall as is desiredfor many practical applications but at the boundary of the steel layerand the fluid.

A number of approaches to this problem have focused on so called“pressure release” at the reflecting wall. In particular, attempts havebeen made to minimise the thickness of the glass reflector or tosubstitute a material with similar acoustic properties to the fluid.However, these approaches are largely impractical or fail in that therequired thickness makes the reflector difficult to fabricate and handleand prone to shatter on mechanical shock or in that a standing wave isnot produced.

Applicant has now surprisingly found that selection of the frequencyand/or the thickness of the reflector in both the λ/2 system and the λ/4systems is of practical value for directing particles to one or morefluid boundary surfaces.

Accordingly, in one aspect, the present invention provides apparatus fordirecting particles entrained in a fluid, comprising a chamber in whicha first wall including means for generating a sound wave of frequency νand a second opposite wall capable of reflecting the sound wave in whichthe first and second walls define a conduit for the passage of the fluidand in which the thickness of the second wall is such that the pathlength of the standing wave in the second wall is a multiple of about ½of the wavelength λ_(r) of the sound wave therein.

It will be understood that the selection of the thickness of the secondwall (or the frequency ν of the sound wave) such that the path length ofthe standing wave in the second wall is about ½ λ_(r), leads to apressure node at one or other or both of the fluid boundary surfaces. Inparticular, it will be appreciated that the second wall resonates at theselected frequency and as a result the relatively high acousticimpedance of the wall in relation to the fluid is effectively reduced tozero. Further, although the energy in the system is focused in theresonating second wall, the magnitude of the force acting on theparticles at the node is still sufficient to drive particles across thedirection of laminar flow in a fluid. In this regard, it observed thatat the selected thickness or operating frequency the heat generated bythe resonance of the second wall leads to only a very small increase inlocal temperature (5 to 10° C.).

The present invention does not require that the thickness of the secondwall or the frequency ν of the sound wave is such that the path lengthof the standing wave in the second wall is a multiple of exactly λ/2 ofthe wavelength of sound therein. In particular, the term “a multiple ofabout ½ λ_(r)”, as used herein, will be understood to mean that the pathlength is within +/−5% of the theoretical value for n (½) the wavelengthλ_(r) of sound in the second wall where n is an even or odd integerincluding 1.

The selection of the thickness of the reflector or the frequency of thesound wave such that the path length of the standing wave is a multiplevalue of ½ λ_(r) must, however, not increase the local temperature tosuch an extent that the speed of sound and, therefore, the wavelength ofthe standing wave in the second wall is significantly changed.Preferably, the value of n ranges from 1 to 5. Still more preferably,the thickness of the second wall is about ½ λ_(r). (n=1)

It will be understood that, subject to the requirement for sufficientenergy in the fluid layer, the thickness of the first wall is notcritical to the present invention. Preferably, the thickness of thefirst wall is an odd multiple (n is an odd integer) of ½ or ¼ of thewavelength λ_(g) of the sound wave therein. Still more preferably, thethickness of the first wall is ½ of the wavelength λ_(g) of the soundwave in therein.

In preferred embodiments of the present invention, the apparatus furthercomprises a material in contact with the first wall, which is capable oftransmitting the sound wave. The inclusion of a coupling layer on theinner surface of the first wall is well known to the art. The thicknessof the coupling layer is, subject to the energy requirement, notcritical to the present invention. However, the preferred thickness isan even or odd multiple (n is an even or odd integer) of ½ or an oddmultiple (n is an odd integer) of ¼ of the wavelength λ_(t) of the soundwave in therein.

Further, the width of the conduit is, subject to the energy requirement,not restricted by the present invention. Preferably, however, the widthof the conduit is an even or odd multiple (n is an even or odd integer)of ½ or an odd multiple (n is an odd integer) of ¼ of the wavelengthλ_(f) of the sound wave in therein. Of course, where the width is ½ or amultiple of ½ of the wavelength λ_(f) of the sound wave in the fluidmore than one pressure node is present in the fluid layer. However, inthis system, a pressure node will always be present at the boundaries ofthe fluid layer. Similarly, where the width of the conduit is an oddmultiple of ¼ of the wavelength λ_(f) of the sound wave in the fluid, aplurality of pressure nodes will also be present. However, in thissystem, a pressure node will always be present at the boundary of thefluid layer with the second wall.

In a highly preferred embodiment of the present invention, however, thewidth of the conduit is a ¼ of the wavelength λ_(f) of the sound wave inthe fluid. In this embodiment a single pressure node is present in thefluid layer and located at the boundary of the fluid layer and thesecond wall. This embodiment is particularly advantageous in that theparticles in the fluid are driven solely to the boundary surface betweenthe fluid layer and the second wall.

As mentioned previously, the energy of the standing wave in the systemof the present invention is an important consideration. Preferably,therefore, the frequency ν, the thickness of the first and second walland the width of the conduit are such that the overall path length ofthe standing wave (i.e. Σ λ_(r), λ_(t), λ_(f), λ_(g)) in the system is amultiple of λ/2. Sufficient energy, at desired operating frequencies andthickness, can be obtained by selection of the amplitude of the soundwave generated by the first wall

In preferred embodiments of the present invention, the material capableof generating an ultrasound wave comprises a piezoceramic. In theseembodiments, the apparatus further comprises means for applying analternating potential to the piezoceramic. The magnitude of the appliedalternating potential controls the amplitude of the sound wave.Preferably, the frequency of the applied potential is at or adjacent thefundamental resonance frequency of the piezoceramic. However, otherfrequencies may also be used. Of course, other sources of ultrasound,including lasers and electrostatic actuators may also be used.

The materials of the transmission layer and the second wall may compriseany such as are known to the art. Preferably, the material capable ofreflecting the sound wave comprises glass. Advantageously, the secondwall comprises a glass microscope slide. The transmission layer maycomprise, in particular, steel, carbon or silicon.

The apparatus according to the present invention may also comprise meansproviding for the flow of the fluid through the conduit. Preferably, themeans comprise a pump or the like.

The present invention allows the manipulation of particles in a fluid toone or other or both of the fluid boundary surfaces. The inventiontherefore offers alternative possibilities for filtering particles fromthe fluid by, for example, collection at one or more of these surfaces.

In preferred embodiments of the present invention, the apparatus furthercomprises means for detecting particles at or adjacent the first and/orsecond walls of the chamber.

In a highly preferred embodiment, the width of the conduit is such thatthe path length of the standing wave therein is a ¼ of the wavelengthλ_(f) of the sound wave in the fluid (a λ/4 system). In this embodimentthe detection means is provided at the second wall since only a singlepressure node, located at the boundary of the fluid and the second wallis present in the fluid layer.

The present invention is not limited by any particular means fordetecting the particles. The apparatus may comprise any suitabledetection means for detecting particles known to the art. Preferably,however, the detection means comprises a biosensor including a sensingmedium capable of sensing particles such as bacteria, viruses, DNA,proteins and the like. Still more preferably, the detection meanscomprise an optical biosensor.

The sensing medium may, in particular, comprise an agarose or dextrangel matrix supporting a capture agent specific for particles ofinterest. Preferred forms of capture agents include antibodies orlectins. Of course the sensing medium may comprise other inert matricesand/or support a plurality of capture agents specific for differentparticles.

In one embodiment, the second wall is, at least in part, provided with alayer of the sensing medium. In particular, the second wall and thesensing means may comprise a surface plasmon resonance (SPR) sensorchip, a metal clad leaky waveguide (MLCW) sensor chip or the like. Suchchips enable the detection of captured particles by a shift in thecoupling angle of light incident the under side of the chip to anevanescent wave in the sensing medium or the fluid. See for example, ourco-pending patent application PCT/GB02/045045 incorporated by referenceherein.

The detection means may further comprise a microscope, a CCD videocamera and/or an image analysis system. Alternatively or additionallythe detection means may comprise means detecting a shift in the couplingangle of light to an evanescent wave in the sensing medium or the fluid.The detection means may, in particular, image the upper surface of thechip or may instead image light scattered or emitted from the particleson the chip.

The detection of the particles does not, however, necessarily requirethat the second wall or chip can be dismantled from the apparatus. Inparticular, it will be apparent that an SPR or MCLW chip permits thatdetection may be carried out directly and even during sonication.Preferably, however, the second wall or chip can be removed from theapparatus so as to minimise vibration effects and/or facilitatedetection by monitoring light scattered or emitted from the sensingmedium of the chip. The removal of the chip advantageously permits itsreplacement and/or substitution.

The concentration of the particles in the sample fluid may promote theformation of aggregates, which may reduce the capture of particles bythe sensing medium. Preferably, therefore, the apparatus includes meansfor optimising the concentration of the particles in the fluid inconjunction with the flow rate and pressure gradient. Such means maycomprise means for filtering or diluting the fluid.

Of course, the selection of the width of the conduit need not be limitedto a λ/4 system. In particular, the apparatus may alternatively comprisea λ/2 system in which a pressure node occurs at both fluid boundarysurfaces. In this embodiment, the detection means may be provided at thepiezoceramic or coupling layer and/or the second wall. Preferably, thedetection means provided on the transmission layer or piezoceramiccomprises a regenerable, biological sensing medium. The regeneration ofthe sensing medium enables multiple use of the chamber without theexpense of providing a new piezoceramic or coupling layer.

Other embodiments comprising multiple λ/4 and λ/2 systems may also beused provided the penalty of a plurality of pressure nodes is acceptablefor the purposes of detection.

In a second aspect the present invention provides a method for detectingparticles in a fluid comprising the steps of i) passing the fluidthrough a chamber comprising a first wall including means generating asound wave of frequency ν and a second opposite wall capable ofreflecting the sound wave which together define a conduit for thepassage the fluid and detection means for detecting particles at thefirst and/or second walls ii) selecting the frequency ν such that thepath length of the standing wave in the second wall is a multiple ofabout ½ of the wavelength λ_(r) of the sound wave therein and iii)detecting the particles.

The chamber may comprise any of the features of the embodiments of theapparatus according to the first aspect of the present invention.Preferably, however, the width of the conduit in the chamber is ¼ of thewavelength λ_(f) of the sound wave in the fluid and detection means isprovided at the second wall.

The optimum capture of particles is dependent on the flow rate of thefluid in the conduit as well as the pressure gradient in the fluid. Theoptimisation of the flow rate will be within the skill of the ordinarypractitioner. Preferably, the flow rate is such that the flow in theconduit is laminar and the residence time of the fluid in the conduitpermits maximum capture of the particles on the sensing medium. Theoptimisation of the pressure gradient and flow rate should, however,preferably avoid the formation of aggregates of the particles, which mayreduce the possibility of their capture at the second wall.

The concentration of the particles in the sample fluid may promote theformation of aggregates, which may reduce the capture of particles bythe sensing medium. The method may, therefore, comprise a preliminarystep of adjusting the concentration of the particles in the fluid.

The detection step may be performed using any suitable technique.Preferably, the detection step relies on an evanescent technique, suchas surface plasmon resonance, and sensing means comprising a biologicalsensing medium on which the particles are captured. A change in thelocal refractive index of the medium on capture of the particles leadsto a shift in the angle of incident light required for resonance.

The method of the present invention may comprise the additional step ofremoving the second wall and sensing medium from the chamber prior tothe detection step. In this embodiment, vibration effects may be avoidedand the evanescent technique may be permit detection of light scatteredor emitted from the sensing medium. Alternatively, or additionally thedetection step may comprise visualisation of particles captured on thesensing medium by a microscope, video camera and/or imaging system or onlight scattered or emitted from captured particles.

The apparatus and method of present invention provide enhanced detectionof particles, and in particular, pathogens such as bacteria or viruses,through ultrasonic deposition on a surface sensor. The invention offersimproved sensitivity over other apparatus in that the ultrasonic forceacting on particles of diameter in the order of 1 μm is able to overcomethe. slow diffusion rate and, in particular, to cause them to cross theparallel flow lines in a fluid in laminar flow. A particular advantageof the invention lies in the fact that the second wall and associatedsensing means can comprise a cheap arrangement of a microscope slide andsensing medium which can be removed and replaced.

The present invention will now be described with reference to a modelstudy, a preferred embodiment and the accompanying drawings in which

FIG. 1 is a schematic representation of the an acoustic chamberaccording to the present invention;

FIG. 2 shows results of the model study in which the distribution ofacoustic pressure and energy in the chamber is related to a number ofalternative configurations of a λ/2 system;

FIG. 3 shows results of the model study in which the distribution ofacoustic pressure and energy in the chamber is related to a number ofalternative configurations of a λ/4 system;

FIG. 4 shows the results of the model study in which the frequency ofthe acoustic energy in the fluid layer is related to the configurationsof FIG. 2;

FIG. 5 shows the results of the model study in which the frequency ofthe acoustic energy in the fluid layer is related to the configurationsof FIG. 3;

FIG. 6 is an exploded view of apparatus according to a preferredembodiment of the present invention;

FIG. 7 shows photographs illustrating the collection of Bacillussubtilis var niger (B. globiggi) spores using the apparatus of FIG. 6;

FIG. 8 shows photographs of illustrating the collection of B. globiggiat various concentration of sample and residence time.

Having regard now to FIG. 1, there is shown an acoustic chamber,generally designated 11, comprising a first, transducer wall 12comprising a piezoceramic and a transmission layer 13 comprisingstainless steel, and a second, reflecting wall 14 also comprisingstainless steel. The first and second walls define a conduit 15 for thepassage of a water layer.

A transfer matrix model, described by Nowotny, H. et al, in J. Acoust.Soc. Am., 1987, 90, 1238-1245, simulating acoustic wave propagation ofthrough the system was executed for a number of configurations of theacoustic chamber. The physical properties of the materials modelled aregiven in Table 1. (The model has recently been experimentally validatedby Hawkes, J. J. et al., ibid. 2002, 111, 1259 and Grölschl, M.Fundamentals Acustica—acta Acustica, 1998, 84, 432-447.).

The positioning of desired pressure nodes between the chamber walls wasexamined. A system of fundamental frequency of 3 MHz and acousticquality factors of 1000 for the different layers was assumed althoughprevious fitted quality factor results were 150 and 350 for the waterlayer and the piezoceramic respectively. Values for pure water is morethan 10,000 and the piezoceramic used are, however, quoted. Thepiezoceramic was assumed to have a source voltage of amplitude 1V and asource resistance of 50 ohms. The model does not take into account theglue layer used to fix the transmission layer to the piezoceramic.

The thickness of the transmission layer and the reflector are modelledat 3 MHz with values that exactly correspond to λ/2 or λ/4 or 0. Thewidth of the conduit or thickness of the water layer was modelled at 3MHz with a value exactly corresponding to ½ λ_(f) and ¼ λ_(f). Thesedimensions are also shown in Table 1 (mm). Results are expected to besimilar for systems in which the thickness of these layers correspondsto multiples of ½ λ and ¼ λ. For example, a ¼ λ layer is expected tobehave in a similar manner to ¾ λ, 5/4 λ etc. layers whilst a ½ λ isexpected to behave in a similar manner to 3/2 λ, 5/2 λ etc. layers.However, increasing the path length of the standing wave in the variouslayers will alter the frequency spectra bringing the peaks closertogether (see FIGS. 4 and 5).

TABLE 1 Piezoce- Steel Water ramic A B, D C Speed of sound/ms⁻¹ 40806100 1500 Mass density/kgm⁻³ 7700 7800 1000 Acoustic quality factor 10001000 1000 Dielectric constant/Fm⁻¹ 1.2 × 10⁻⁸ Tangent of dielectric lossangle 3.0 × 10⁻³ Electromechanical coupling factor 0.48 Electrodearea/mm² 200 Thickness at 3 MHz (Q) 0.508 0.125 Thickness at 3 MHz (H)0.680 1.016 0.250

Referring now to FIGS. 2 to 6, the large number of configurationsexamined are referred according to a terminology expressing the pathlength of the standing wave in each layer at 3 MHz as zero (0), aquarter (Q) and half (H) of the wavelength of sound therein. Thethickness of the piezoceramic was modelled at a half wavelengththroughout and is not therefore referred to in the Figures. It will beapparent that the expression “0HQ” refers to a configuration in whichthere is no transmission layer, and the thickness of the water layer andreflector correspond respectively to a path length of a half wavelength(λ_(f)0.25 mm) and a quarter wavelength (λ_(r) 0.508 mm).

The model was examined for the position of a pressure node in the waterlayer at one or other or both of the fluid boundary surfaces. Referringnow to FIG. 2, there are shown results for the spatial distribution ofacoustic pressure (left-hand side, kPa) and energy density (right-handside, J/m³) for all nine possible configurations of a λ/4 system (0.125mm, thickness shown along abscissa in mm). The vertical lines indicatethe boundary positions in each system.

As may be seen (left-hand column) the absence of the second wall leads,as may be expected, to a pressure node at the boundary of the fluidlayer with air in this system. The absence of the second wall means thatthese three configurations are of no real practical value for thedetection of particles. A reflector of thickness providing a path lengthof ¼ λ_(r) (middle column) leads to a pressure node at the boundary ofthe fluid layer and the transducer. However, a reflector of thicknessproviding a path length of ½ λ_(r) (right-hand column) leads to apressure node in the fluid at or adjacent the reflector surface.

Referring now to FIG. 3, there are shown results for the spatialdistribution of acoustic pressure (left-hand side, kPa) and energydensity (right-hand side, J/m³) for all nine possible configurations ofa λ/2 system (0.25 mm, thickness shown along abscissa in mm). Thevertical lines indicate the boundary positions in each system.

As may be seen (left-hand column) the absence of the second wall leads,to an additional pressure node at the boundary of the transducer and thewater layer compared to λ/4 system. A reflector of thickness providing apath length of ¼ λ_(r) (middle column, prior art) leads to a node in themiddle of the water layer. However, a reflector of thickness providing apath length of ½ λ_(r) (right-hand column) leads to a pressure node inthe fluid at or adjacent the reflector and transducer.

The model shows, therefore, that for configurations in which there is noreflector or a reflector providing a path length of ½ λ_(r), (xy0 or xyHconfigurations) there is always a pressure node at the outer boundary ofthe water layer. Further, the model shows that there is an additionalpressure node at the lower boundary of the water layer forconfigurations in which the water layer provides a path length of ½λ_(f) (xH0 or xHH configurations). A pressure node in the middle of thewater layer only occurs for the combination of a water layer providing apath length of ½ λ_(f) and a reflector providing a path length of ¼λ_(r).

As may be seen from the pressure scales in FIGS. 2 and 3, the acousticpressures obtained are particularly high where the total acoustic pathlength of the system is a multiple of λ/2 (0QQ, QQ0, QQH, HQQ and 0H0,0HH, QHQ, HH0, HHH).

The acoustic energy density in the water layer is, however, alsodependent on the thickness of the reflector. For a reflector ofthickness of ½ λ_(r), the acoustic energy is much higher in thereflector than in the water layer because for these configurations thedisplacement amplitudes have the same maximum value in both layersadjacent the water layer. This follows from the condition of continuityfor the displacement that must apply at the water-reflector interface.

Referring now to FIGS. 4 and 5, the frequency spectra of the acousticenergy density in the water layer are shown for the systemconfigurations of FIGS. 2 and 3 respectively. As may be seen, a maximumacoustic energy in the water layer is obtained for a piezoceramicresonance frequency of 3 MHz for all configurations in which the totalacoustic path length in the system is a multiple of λ/2. It will,however, be noted in this regard that the energy in the figures are notwholly indicative of the efficiency of a particular configuration inthat the energy density is also a function of the applied voltage andthe total electrical input into the system. Nonetheless, it is clearthat the best configurations are 0QQ, QQ0, QQH, HQQ and 0H0, QHQ, HH0.

Taken together the results of the model suggest that the most efficientconfiguration for directing particles to the reflector is QQH. Althoughthe results for 0QH and HQH look very similar to those obtained for 0QQand HQQ respectively (total acoustic path length a multiple of λ/2), thesimilarity is apparently due to a smoothing effect caused by the 50 ohmsource resistance. The effect is revealed by the fact that the energydensity spectrum for a zero source resistance for the configuration 0QH,for example, is a curve with a double peak (not shown). The double peakis characteristic of configurations in which the total acoustic pathlength at 3 MHz is not a multiple of λ/2.

Other configurations not fulfilling the path length criterion show adouble peak near 3 MHz (0Q0, HQ0, 0HQ or HHQ) or peaks far below andabove 3 MHz (QQQ. QH0). However, the configurations 0HH and HHH showonly a very small peak at 3 MHz as well as two larger peaks eithereither side even though they fulfil the path length criterion at 3 MHz.The effect would appear to be attributable to the smoothing effect ofthe source resistance which at 3 MHz is large compared to the resonatorimpedance.

The model provides similar results for pressure node positions andfrequency spectra for a transmission layer and reflector consisting ofcarbon (speed of sound 4260 m/s, mass density 1470 kg/m³) or silicon(speed of sound 8430 m/s, mass density 2340 kg/m³). The model alsosuggests that a change in the path length of the sound wave in thereflector from ¼ λ_(r) to ½ λ_(r) (especially in configurations 0HH andHHH) can lead to a movement of particles from the centre to the walls ofthe chamber. Further, particles may also be moved from one wall toanother. The model further suggests that it is advantageous to operateat the fundamental resonance of the piezoceramic. However, otheroperating frequencies are not necessarily excluded and may produce someuseful systems.

The model was experimentally verified using a system of configurationQQH. Referring now to FIG. 6, there is shown an acoustic chambercomprising an electrode 12 formed by a plane ultrasonic transducer(PZ26, 30 mm square, 3 MHz thickness resonance, Ferroperm, Denmark) onwhich a stainless steel coupling plate 13 (1.5 mm, 3λ_(t)/4 at 3 MHz) isglued. The back of the electrode 12 is etched to give a central 20 mm by10 mm transducer area.

An opposing plane glass acoustic reflector 14 comprising an antibodycoated glass microscope slide (1.0 mm, antibodies specific for B.globiggi) is held in place by a brass shim spacer 16 and a brasstop-plate 17 secured with screws 18. The brass shim spacer 16 (0.125 μm)is arranged between the coupling plate 13 and the glass reflector 14 andcomprises an aperture 19 providing a window or field of view (14 mm by64 mm). A silicon rubber gasket (not shown) located within the window 19defines a conduit (10 mm×60 mm) for the passage of fluid and forms anair and water seal between the slide 14 and the coupling plate 13. Thechamber is fixed to a Perspex® base 20 by screws 18. The base 20 isprovided with an inlet 21 and outlet 22 for the passage of the sampleand a connector 23 for applying a potential to the transducer. Aperistaltic pump (not shown, Gilson minipuls 3) is arranged to pumpsuspensions of B. globiggi into the channel. The generation and controlof the frequency and voltage applied to the transducer is performedaccording to Spengler J. F. et al., Bioseparation, 2001, 9, 329-341 andHawkes J. J. and Coakley, W. T., Sensors and Actuators, 2001, B, 75,213-222).

The antibody coated microscope slide was prepared by soaking apre-silanated slide (SILANE-PREP™, Sigma) in 1% glutaraldehyde solutionin de-ionised water over 1 to 3 hours. The slide was washed withphosphate buffered silane (PBS, five times) and dried in a stream ofnitrogen. A 100 ug/ml polyclonal rabbit α-BG antibody solution in PBS(200 μl) was applied to a central region of the slide (10 mm by 20 mm).After standing for 3 hours the slide was washed with a PBS solutioncontaining 0.05% Tween™ and the excess solution drained.

The area of the antibody coated region of the slide covers a greaterarea than etched area of the transducer enabling deposition also to beexamined upstream and downstream of the ultrasound field. The field ofview was examined during the experiment or following removal of theslide using a video camera and/or microscope.

EXAMPLES

Spore suspensions of B. globiggi 24 were prepared by dilution of aconcentrated solution of 2×10¹⁰ c.f.u. ml⁻¹ in PBS (pH 7.4). Dilutionsof 1×10⁸ to 1×10⁵ cfu ml⁻¹ were confirmed by haemocytometer count priorto use.

A Polystyrene latex bead (2.8 μm) suspension in water (10% w/v, PolymerLaboratories, UK) was diluted in distilled water to a concentration of8×10⁶ beads ml⁻¹.

Example 1

A frequency scan for the latex bead solution showed that the optimalfrequency for driving the particles to the reflector surface was 2.915MHz for an apparatus (A) in which the channel width was 178 μm. Theoptimal frequency was 2.882 Hz for an apparatus (B) in which the channelwidth was 200 μm. The path length of the standing wave in the waterlayer in these systems lies between a λ/4 and λ/2 wavelength of thespeed of sound in water. These frequencies were employed in subsequentexperiments.

Example 2

Apparatus A

A suspension of spores (1×10⁸ cfu ml⁻¹) was passed through the apparatusat a flow rate of 2 ml/min. In the absence of ultrasound the spores wererandomly distributed across the field of view with an even concentrationthroughout the suspension. No contact of the spores with the reflectorwas identified. However, when the ultrasound was switched on (0.8V_(rms) applied to the transducer) spore clumps formed in the region ofthe ultrasound field within one second. The spores remained stationaryagainst the flow and the size of the clumps increased with increasedresidence time in the field. Removal of the slide from the apparatusshowed that only bonded spores remained attached.

The spore deposition to the reflector surface during ultrasonicationshowed a regular pattern of areas of adherence in which the spacingbetween the areas appeared to correspond to the half wavelength of soundin water (0.26 mm at 2.90 MHz). This result suggests the possibility ofachieving deposition on discrete areas of the slide. Within each areathe spores adhered to 40 to 95% of the available surface area. Thegeneral position of spore clumps in the chamber did not change duringultrasound exposure. Further experiments focused on these areas.

Referring now to FIG. 7, the deposition of the spores (1×10⁸ cfu ml⁻¹)in relation to specific regions of the chamber in the field of view areshown. Little or no deposition occurred before the spores reached thearea of the ultrasound field (FIG. 7 a), whereas maximum depositionoccurred within the field (FIGS. 7 b edge and c centre). A reducednumber of spores adhered to the antibody layer beyond the field (FIG. 7d).

Example 3

The applied voltage to the piezoceramic was varied. At 0.2V_(rms) spore(1×10⁸ cfu ml⁻¹) deposition in apparatus A was negligible compared todeposition at 0.5V_(rms). Experiments in this apparatus were continuedat 0.8V_(rms). Similar observations for apparatus B led to the selectionof 3.5 V_(rms).

Example 4

Apparatus B

Referring now to FIG. 8, spore deposition to the antibody coated slidewas compared after ultrasound exposure (3.5 V_(rms) at a flow rate of0.4 ml/min) of samples of concentrations and 6.6×10⁶ cfu ml⁻¹ for 3 and30 min and removal of the slide from the apparatus. During exposure thedeposition steadily increases (FIG. 8 a, 0 min, 8 b, 3 min and 8 c 30min).

Example 5

Apparatus B

The lowest concentration at which spores were visible under a microscopeon a slide removed from the apparatus after ultrasound was determined tobe 3.3×10⁵ cfu ml⁻¹. The figure represents a 200-fold increase insensitivity compared to detection in which ultrasound is not used.

The experimental results confirm the prediction of the model thatultrasonic manipulation of particles to a boundary surface is possible.In particular, they confirm that the acoustic force acting on particlesis sufficient to move particles at right angles to the direction oflaminar flow. The experiments also confirm, in view of the similarity ofthe results obtained with both apparatus (in which the variation inwidth of conduit is about 10%) that optimum movement does not depend onthe path length of the standing wave in the fluid layer.

Improved experimental results providing greater sensitivity (to sporeconcentrations of about 1×10⁴ cfu ml⁻¹) are envisaged through optimaldesign of the reflector leading to better resonance.

1. Apparatus for directing particles entrained in a fluid, comprising achamber having a first wall, including means for generating a sound wavehaving a frequency v, and a second, opposite wall capable of reflectingthe sound wave in which the first and second walls define a conduit forthe passage of the fluid, and in which the thickness of the second wallis such that the path length of the standing wave in the second wall isa multiple of about ½ the wavelength λ_(t) of the sound wave therein. 2.Apparatus according to claim 1, in which the first wall furthercomprises a coupling layer.
 3. Apparatus according to claim 2, in whichthe width of the conduit is a multiple of ½ or ¼ for the wavelengthλ_(f) of the sound wave in the fluid.
 4. Apparatus according to claim 3,in which the thickness of the material transmitting the sound wave inthe first wall is a multiple of ½ or ¼ of the wavelength λ_(t) of thesound wave therein.
 5. Apparatus according to claim 1, in which thethickness of the material capable of generating the sound wave is an oddmultiple of ½ of the wavelength λ_(g) of the sound wave therein. 6.Apparatus according to claim 1, in which the total acoustic path lengthof the wave is a multiple of ½ of the wavelength of the sound wave λtherein.
 7. Apparatus according to claim 1, in which the materialcapable of generating the sound wave is a piezoceramic.
 8. Apparatusaccording to claim 7, in which the frequency v of the sound wave is ator adjacent the resonant frequency of the piezoceramc material. 9.Apparatus according to claim 1, in which the second wall comprisesglass, steel, carbon or silicon.
 10. Apparatus according to claim 1, inwhich the material in the first wall capable of transmitting the soundwave comprises steel, carbon or silicon.
 11. Apparatus according toclaim 1, in which the sound wave is an ultrasound wave.
 12. Apparatusaccording to claim 1, further comprising detection means for detectingparticles at or adjacent the first and/or second walls.
 13. Apparatusaccording to claim 12, in which the detection means comprise abiological sensing medium.
 14. Apparatus according to claim 13, in whichthe sensing medium comprises one or more antibodies or lectins. 15.Apparatus according to claim 12, in which the second wall is removable.16. Apparatus according to claim 12, in which the second wall and thesensing medium comprise a surface plasmon resonance or a metal leakywaveguide chip.
 17. Apparatus according to claim 16, in which thedetection means further comprise means providing light incident thesecond wall and means detecting a change in the angle thereof requiredfor resonance or optical coupling.
 18. Apparatus according to claim 16,in which the detection means further comprise means detecting lightscattered or emitted from the particles.
 19. A method of detectingparticles in a fluid comprising the steps of i) passing the fluidthrough a chamber comprising a first wall including means for generatinga sound wave of frequency v and a second, opposite wall capable ofreflecting the sound wave which together define a conduit for thepassage of the fluid and detection means for detecting particles at thefirst and/or second walls, ii) selecting the frequency v such that thepath length of the standing wave in the second wall is a multiple ofabout ½ of the wavelength λ_(t) of the sound wave therein and iii)detecting the particles.
 20. A method according to claim 19, in whichthe width of the conduit is a ¼ of the wavelength λ_(f) of the soundwave in the fluid.
 21. A method according to claim 19, in which thedetection step is preceded by the removal of the second wall from thechamber.